Brefeldin A (NSC 89671), a macrolide isolated from Penicillium brefeldianum, is undergoing preclinical evaluation as an antitumor agent at the National Caner Institute. A specific assay for Brefeldin A was developed involving its extraction from plasma into diethyl ether followed by derivatization with heptafluorobutyrylimidazole prior to gas chromatography with electron capture detection. Both secondary hydroxyl groups of Brefeldin A were rapidly and quantitatively esterified. The lowest concentration of Brefeldin A determined with acceptable reproducibility in 50 fl of plasma was 0.1 fg/ml. This assay has been utilized to investigate Brefeldin A plasma pharmacokinetics in mice. Brefeldin A plasma levels declined in an apparent biphasic manner during 60 min after bolus i.v. injection. Due to the extremely short initial disposition phase, the terminal phase was the primary determinant of Brefeldin A disposition, accounting for 74% of the area under the plasma profile. The central compartment (0.71 l/kg) and total body (1.5 l/kg) apparent volumes of distribution were low, but indicative of drug distribution into peripheral tissues. Brefeldin A was not bioavailable following p.o. administration. Therefore, long term exposure to therapeutic concentrations of Brefeldin A (> 0.1 fg/ml) may require a continuous infusion schedule. Further, interest in Brefeldin A has grown to such proportions that a continuous supply of the compound is now in demand. The structural complexity of Brefeldin A precludes facile synthesis, thus large scale isolation from Penicillium brefeldianum was investigated. Optimization of yield and assessment of purity of Brefeldin A from fermentation broths were thus guided by application of the developed assay.